Microfluidic device and method of manufacturing the microfluidic device

ABSTRACT

A microfluidic device having a substrate with an array of curvilinear cavities. The substrate of the microfluidic device is preferably fabricated of a polymer such as polydimethylsiloxane. The microfluidic device is manufactured using a gas expansion molding technique.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 13/304,843, filed on Nov. 28, 2011 and entitled “MicrofluidicDevice and Method of Manufacturing the Microfluidic Device,” and U.S.patent application Ser. No. 12/139,797, filed on Jun. 16, 2008 andentitled “Microfluidic Device and Method of Manufacturing theMicrofluidic Device” and claiming priority to U.S. Provisional PatentApplication Ser. No. 60/929,128, filed Jun. 14, 2007, all of which arehereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to microfabrication, and moreparticularly, to microfabrication of a microfluidic device.

BACKGROUND

Microfabrication technology is used to manufacture microfluidic devicessuch as lab-on-a-chip systems (“LOC”), which separate or mix fluids andperform biochemical reactions using the separated or mixed fluids.Microfluidic devices are also used to sort cells and provide a means toconduct single cell assays. Microfluidic devices include a substrate onwhich channels and chambers are formed. Soft lithography methodsemploying polymer-molding to generate LOC devices has enabledconsiderable innovation in the application of microfluidic devices forcell sorting and microcell culture. Precise control over laminar flowstreams has enabled the selective spatial exposure of bioactive agentsto cells and the investigation of mechanotransduction and cell responseto shear stress under laminar or pulsatile flows. Precise control oversolutions applied to the device enable control over the device functionand application. Many challenges exist however, to creating a technologyplatform that can sort cells from a diverse population, maintain them inculture, uniformly direct their fate (e.g., differentiation,elimination) and interrogate cell signaling responses in situ as afunction of cell density.

Parallel-plate microchannel systems employing physical features(pillars) and adhesive interactions have proven useful in cellseparation. The pillars are generally fabricated by a reverse moldingtechnique. FIG. 1 is directed to a prior art method 10 used to fabricatepillar arrays. A silicon wafer mold 12 is provided. Deep reactive ionetching (DRIE) is used to make pits 14 in silicon wafer mold 12. Ahydrophobic material is coated onto silicon wafer mold 12 for easyremoval of a polymer after it has been cured. A common polymer used inmicrofabrication of microfluidic devices is polydimethylsiloxane (PDMS).PDMS 16 is cast onto the hydrophobic silicon wafer mole 12 as shown inStep (a). In Step (b), the standard practice is to apply a vacuum todegas PDMS 16 and deplete trapped gas, for example, air, nitrogen,helium and/or argon, in pits 14 and dissolved in PDMS 16. PDMS 16 isthen cured for example at 100° C. for two hours as shown in Step (c).The reverse molded pillar array 18 is removed from silicon wafer mold12.

FIG. 2 shows a pillar array for use in cell separation. The disadvantageof pillar arrays is that, over time, as the adherent cell number builds,the microchannel hydrodynamic resistance and flow velocity can change.Devices can become clogged.

It is a primary object of the invention to provide a microfluidic devicethat can integrate cell sorting, microcell culture and real timediagnostics. It is another object of the invention to provide amicrofluidic device that is applicable for use in diagnostic,therapeutic and investigative research, particularly in the areas ofsorting rare cells and investigating stem cell and cancer cell biology.It is a further object of the invention to provide a facile andeffective method of manufacturing microfluidic devices incorporatingstructures with novel geometries.

SUMMARY

These and other objects and advantages are accomplished by amicrofluidic device. In one embodiment, the microfluidic device has asubstrate with a plurality of curvilinear cavities. Each curvilinearcavity has an inner surface that curves outward from a rounded bottomlocated at a point furthest from the opening of the cavity to a maximumdiameter, and then curves inward from the point of the maximum diameterto the opening. The maximum diameter of the inner surface is variablebut the preferred embodiment has a maximum diameter of the inner surfacegreater than the diameter of the opening. In one embodiment, the ratioof the maximum diameter to the diameter of the opening is greaterthan 1. The maximum diameter can be, for example, approximately 60 to200 microns, and the diameter of the opening can be, for example,approximately 150 to 350 microns. The curvilinear cavities lack an edgeother than the opening, and can be spherical, oblong, oval or any othercurved shape.

In one embodiment, the curvilinear cavities are provided in a variety ofdifferent arrays such as in evenly spaced rows or in staggered rows. Thecavities may be all of the same size, all of the same shape, of variedsizes, and/or of varied shapes. The cavities may be spaced at a distancein a range of about two times the diameter of the opening of thecavities to about ten times the diameter of the opening of the cavities.The cavities may be fused to form of a linear tubular cavity or obtuseshaped cavity.

In another embodiment, the substrate of the microfluidic device ispreferably fabricated of a polymer such as polydimethylsiloxane (PDMS).Other examples include, but are not limited to, polysiloxanes, andcarbon-based polymers including polyacrlyamides, polyacrylates,polymethacrylates or mixtures thereof.

In yet another embodiment, the cavities comprise a coating for selectivecapture of cells. The coating may provide a microenvironment beneficialfor the culture of specific cells. The coating may comprise protein orbiochemicals. The coating may be deposited by vacuum-assisteddeposition. Examples of the coating include, but are not limited to,antibodies (IgG, IgM, IgE etc), selectins, collagens, fibronectins,laminin, cytokines, chemoattractants, signaling molecules, antigens,receptor ligands, biochemical agonists and antagonists, and/or inorganiccompounds including silanes and/or other homo- or hetero-functionalagents.

In yet another embodiment, the microfluidic device may have one or moresensors embedded therein. The sensors may be embedded in the substratebelow the cavity or in the cavity, itself. An example of a sensor is aporous silicon optical sensor.

In another embodiment, a method of manufacturing a microfluidic deviceusing a gas expansion molding (GEM) technique is provided. A mold isfabricated to provide one or more pits therein. A polymer premix isapplied onto the mold, covering the one or more pits to create aninterface between the polymer layer and the mold. The mold with saidpolymer premix layer thereon sits for a period of time at roomtemperature to allow the premix to self level and to allow dissolvedgas, for example, air, nitrogen, helium, and/or argon to rise to the airpremix interface. When the polymer is cured at high temperature the gasdissolved in the premix and trapped in the one or more pits expands. Gastrapped in the pit forms a meniscus in the polymer premix. The curingprocess may allow gas trapped in the polymer to diffuse. The gas maycombine with gas trapped in one or more pits. As the polymer is cured,the gas at the polymer-mold interface expands to create microbubbles.Residual gas trapped in the polymer diffuses to enhance bubble growth.The cured polymer has cavities formed therein where the microbubbles hadformed. The cavity size and shape depends on the mold parameters andprocess conditions. The polymer substrate having the cavities isseparated from the wafer for use as a microfluidic bio or chemicalreactor.

In a further embodiment, the mold may be a silicon wafer with pitsformed by deep reactive ion etching (DRIE) or the mold may be amicrobubble array formed in PDMS. The mold may be any material with deeppits that can maintain its shape at high temperature and isintrinsically hydrophobic or rendered hydrophobic by a coating process.

In a further embodiment, the step of allowing the mold with the polymerlayer thereon to sit is conducted for about 10 to about 60 minutes at ahigh temperature in the range of about 20 to about 200° C. A preferredcuring temperature range is from about 65 to about 100° C.

In yet a further embodiment, the polymer is applied at a thickness inthe range of about 0.1 to about 5000 microns. The polymer may be curedat a temperature in the range of about 20 to about 200° C.

In another embodiment, the mold is coated with a hydrophobic materialprior to the step of applying the polymer layer onto the mold. Examplesof hydrophobic materials include, but are not limited to, silane andfluorinated polymer coatings, such as Teflon. The coating may beproduced by gas plasma deposition or chemical surface functionalization.The chemical surface functionalization may use alkoxy coupling agentscomprising silanes, titanates, zirconates and zircoaluminates. Examplesof silanes include, but are not limited to, 1H- or2H-perfluoro-decyltrichlorosilane. A preferred coating is a Teflon-likefluoropolymer produced by plasma processes.

In yet a further embodiment, the pits in the mold are provided in anarray comprising pits in evenly spaced rows, pits in staggered rows,pits of the same size, pits of the same shape, pits of varied sizes;and/or pits of varied shapes. The shapes of the pits include, but arenot limited to, polygonal, circular, oval, and oblong cross-section. Thepolygonal cross-section may include, but is not limited to, circular,triangular, square, rectangular, hexagonal or octagonal cross-section.

In a further embodiment, the pits may have a depth in the range fromabout 25 microns to about 500 microns. A preferred depth is in the rangeof from about 50 microns to about 150 microns.

In a further embodiment, the pits are positioned at a distance to createseparate microbubbles or curvilinear cavities. The pits may be spaced ata distance in the range of about two times the diameter of the openingof the pit to about ten times the diameter of the opening of the pit.The pits may be spaced at a distance in the range of about 50 to about500 microns.

In still another embodiment, the pits may be positioned at a distance tocreate fused microbubbles or curvilinear cavities. The fusedmicrobubbles or curvilinear cavities may create a single, linear tubularcavity.

In yet another embodiment, the cavities may be coated with a protein orwith biochemicals for selective capture of cells. The coating may beapplied by vacuum-assisted deposition. The coating may comprise but isnot limited to antibodies (IgG, IgM, IgE etc), selectins, collagens,fibronectins, chemoattractants, signaling molecules, antigens, ligands,and/or biochemicals.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be more fully understood and appreciated byreading the following Detailed Description in conjunction with theaccompanying drawings, in which:

FIG. 1 is a schematic diagram showing manufacturing steps of a pillarsubstrate used in prior art microfluidic devices;

FIG. 2 is a micrograph of a reverse molded pillar array formed byconventional vacuum degassing techniques;

FIG. 3 is a schematic diagram of a substrate formed in accordance withthe present invention;

FIG. 4 is a schematic diagram showing manufacturing steps of a substrateformed in accordance with the present invention;

FIG. 5 is a series of micrographs of microbubbles, microcavities, orcavities formed during the GEM technique;

FIG. 6 is a micrograph of microbubbles, microcavities, or cavitiesformed during the GEM technique shown from an angular view;

FIG. 7 is a series of micrographs of various arrays of microbubbles,microcavities, or cavities formed in accordance with the presentinvention;

FIG. 8 is a micrograph of the openings into the microbubble,microcavities, or cavities illustrating the different shapes withmicrobubbles formed thereon;

FIG. 9 is a series of optical micrographs of mutant microbubbles formedusing the GEM technique, illustrating the effect of pit opening andspatial arrangement thereof;

FIG. 10 is a diagram produced by finite element analysis of gasdiffusion in a polymer structure containing preexisting bubbles;

FIG. 11 is a micrograph of a microbubble showing cells cultured in 6days;

FIG. 12 is schematic diagram of a microbubble being coated usingvacuum-assisted technology; and

FIG. 13 is a schematic diagram of a microfluidic device with anintegrated microbubble device and an optical Porous Silicon (PSi)sensor.

FIG. 14 is a graph of the diameter of microbubbles formed in PDMS versusthe diameter of the opening;

FIG. 15 is a graph of: (A) a COMSOL simulation depicting the effect ofresidual gases available to constricted versus unconstrictedmicrobubbles; and (B) the effect of PDMS curing temperature on thediameter of unconstricted (▪) vs. constricted (▴) microbubbles and thecorresponding microbubble formation efficiency, o and x, respectively(n=20);

FIG. 16A is a graph showing normalized microbubble diameter measuredafter regassing the degassed PDMS pre-polymer with nitrogen, argon,carbon dioxide, helium, and compressed air at various aeration times;

FIG. 16B is a graph of the microbubble formation efficiency (MBFE) fornitrogen, argon, carbon dioxide, helium, and compressed air at 0.5, 2,and 10 min of aeration time;

FIG. 17A is a picture of a single HaCaT cell seeded in a plasma-treatedhydrophilic microbubble, where cell spreading and proliferation areevident at 24 hours and epithelial sheets are evident at 72 hours (whitearrows point to cells and red arrows point to some of the spread cells);

FIG. 17B is a picture of a single HaCaT cell was seeded in an untreatedhydrophobic microbubble, where no proliferation or spreading is seen(even at 120 hours);

FIG. 18 is: A) a picture of bright field images of YUSIK metastaticmelanoma cells grown on tissue culture plate (“TCP”) adopt a spreadmorphology; and B) a picture of bright field images of YUSIK metastaticmelanoma cells grown on planar PDMS do not spread but proliferaterapidly in 3D aggregates;

FIG. 19 is a graph of MTT assay results show the effect of the PDMSsubstrate on HaCaT cell proliferation relative to TCP (P-values <0.05and <0.01 are indicated by (*) and (**), respectively);

FIG. 20 is a group of bright field images of YUSIK metastatic melanomacells following nine days of culture in microbubble wells, with focus atthe microbubble square opening (left) and at the bottom of themicrobubbles (right);

FIG. 21 is a graph of COMSOL simulation results contrasting the verticalconcentration profile through microbubble and rectilinear wells as afunction of well depth (y-position=0 corresponds to the well opening)for three microbubble aspect ratios;

FIG. 22 contains pictures of HaCaT cells cultured as a function of time:(A) on untreated planar PDMS and (B) in an untreated hydrophobicmicrobubble (100 μm dia. square opening, 250 μm microbubble diameter),where cells were seeded at near equivalent density, ˜1×10⁴ cells/cm²(scale bars are 150 μm in length);

FIG. 23 is a graph of the diameter of microbubbles over time duringcuring at 100° C.;

FIG. 24 is a collection of images of microbubbles formed over time;

FIG. 25A is a plot of the average microbubble diameter formed atdifferent locations with circular 100 um diameter openings as a functionof PDMS thickness, including a schematic of a 4-inch microbubble arraydefining inner, middle and outer regions that were analyzed; and

FIG. 25B is a graph showing that co-variance as a function of PDMSthickness (0.5 to 4 mm) was <5% (>200 microbubbles counted per datapoint).

DETAILED DESCRIPTION of EXEMPLARY EMBODIMENTS

As will be appreciated, the embodiments of the present invention includea microfluidic device and a method of manufacture thereof. Themicrofluidic device is very useful for cell sorting, microcell cultureand diagnostics in a single integrated device.

Reference is made to FIG. 3, which shows a substrate 30 formed inaccordance with one embodiment of the invention, which substrateprovides a microbubble array that is useful as a microfluidic device.Substrate 30 has two spherical cavities 32 formed therein. Substrate 32is formed by a gas expansion molding (GEM) process 40 depicted in FIG.4. A mask or mold 42 fabricated of Si or other similar material ismanufactured by etching using the DRIE or similar process to producepits 44 therein. The mold may also be made of a polymer materialcontaining pits or deep features. The pits or features 44 may be anyshape or form, including but not limited to square, triangular, circularand rectangular in cross-section. Pits 44 may vary in diameter dependingon the desired use of the end product, preferably in the range fromabout 20 to about 2000 microns, and more preferably in the range ofabout 60 to about 100 microns. By combining large (greater than 500microns) with small (less than 200 microns), pit openings in compositedevices can be formed consisting of reverse and bubble moldedstructures. Inconsistent GEM structures form with pit openings less thanabout 60 microns using conventional polymeric materials, such aspolymeric materials having a low modulus between 1.5 and 2.0 MPa. Anexample of one such material is polydimethylsiloxane (PDMS).

During the etching process, it is possible to produce a hydrophobiccoating on the surface of the silicon wafer mold 42 through the cyclicreaction of the etchants used, for example, SF₆, followed by C₄F₈passivation steps. Alternatively, a hydrophobic agent such as a silanereagent, (e.g., perfluorododecyl-1H-triethoxysilane) or plasmadeposition of a Teflon-like coating may be used to produce a watersurface contact angle in the range of from about 100 to about 150°.Depending on the treatment used, the mold may be immersed in a watersolution for a period of time and immediately dried under a stream of N₂gas for cleaning purposes. The use of a hydrophobic coating is importantfor the easy removal of the polymeric substrate in the final stage ofthe process.

In step (a) of the process 40 shown in FIG. 4, a polymer 46 such as PDMSis cast over the hydrophobic mold and the sample is allowed to settle atroom temperature for a period of time, for example, 30 minutes, to allowtrapped gas bubbles to rise to the liquid/gas interface (Step b). Otherpolymeric material useful for substrates include, but are not limitedto, inorganic polysiloxane (e.g., —Si—O—Si—O—) polymers, organic carbonbased (e.g., —CH2-CHR—) polymers including, but not limited to,polyacrylamides, polyacrylates, and polymethacrylates.

A critical step in this process is the absence of a degassing step. Nodegassing is performed, as required in conventional reverse moldingtechniques. Polymer 46 is cured for a period of time, which time mayvary depending on the polymer used. In this example, PDMS was used andcuring occurred over a period of about two hours at about 100° C. Thishigh temperature causes expansion of the gas in pits 44. A meniscus 48is formed over each pit 44 and serves to nucleate further bubble growthas trapped gas diffuses through the PDMS 46 to form microbubbles 50(Step (c)). The cured PDMS 46 is removed from wafer mold 42 to reveal asubstrate 52 having spherical cavities 54 formed therein as shown inStep (d).

FIG. 5 shows variations in size of spherical cavities 60, 62 and 64created from the microbubbles formed over a square pit opening duringthe curing process of the polymer. The final shape and size of the finalcavities may depend on a variety of factors including, but not limitedto, the thickness of the polymer layer poured over the wafer mold andthe dimensions of the pits in the wafer mold. The thicker the polymerlayer applied onto the wafer mold, the bigger the microbubble created,which is consistent with more residual gas. Keeping the depth of the pitconstant, the size of the bubble increases as the area of the openingincreases, i.e., the size of the bubble created is proportional to thesize of the opening of the pit. FIG. 6 shows a series of curvilinearcavities 66 disposed in a substrate, viewed from an angled perspective.

Spacing of the pits, also known as mask openings, is another nonobviousfeature that must be considered when designing the spherical bubblearray of this embodiment of the present invention. FIG. 7 depicts avariety of arrays that were produced from different arrays of circularmask openings of equivalent diameter. FIG. 7(a) depicts an array ofmicrobubbles 70 formed over evenly spaced pits formed in a 10×10 array.Microbubble openings 72 are all the same diameter as the pit opening,but microbubbles 70 differ in size depending on their spatial locationin the array. Growth of microbubbles located at array corners is lessconstrained in FIGS. 7(a) and 7(c), and also along the outer rows inFIG. 7(b), which shows microbubbles in a staggered array. FIG. 7(c),showing alternating row array, in addition to the larger microbubbleslocated in the corners, also shows larger microbubbles in the end rowsrunning longitudinally. The end rows running laterally are slightlylarger than the microbubbles located interiorly, but smaller than thecorners and end rows running longitudinally. While not wishing to bebound by any theory, it is thought that this phenomenon is related tothe depletion of trapped gas in the polymer premix that can diffuse tothe expanding bubble. FIG. 8 shows microbubble arrays with variousshaped microbubble openings 74 (square), 76 (round), and 78(triangular). These were formed over pits with equivalent size andshape.

Pit openings that are spaced close to each other, such as less than oneor two diameters apart, may produce mutant bubbles 82, as shown in FIG.9. During the polymer cure process, expanding bubbles can fuse formingobtuse shapes depending upon the spatial alignment of pit openings. Itis also possible that the polymer film between adjacent microbubbleopenings is torn off during the mold separation process creating a muchlarger microbubble opening. Mutant bubbles exhibit unique flowproperties. One example of the formation of mutant bubbles has beenfound using pit openings greater than about 40 microns in diameter withpolymer thicknesses of about 0.5 to about 5 mm.

FIG. 10 is directed to a micrograph created using finite elementanalysis. As noted above, it has been determined that close packed pitsproduce smaller microbubbles whereas isolated pits (e.g., inter-pitspacing greater than about 5× to 10× the pit diameter) produce largerbubbles. While not wishing to be bound by any theory, it is thought thatrapid expansion of gas in the pit nucleates a meniscus and bubble growthrecruits trapped gas in the polymer. Competition for gas in the polymeris a limiting factor in bubble growth during the cure process.

In FIG. 10, preliminary simulations were conducted to determine if theproblem of vapor bubble growth is tractable. The initial conditionspositioned pre-existing bubbles in a polymer containing dissolved gasabove a pit. Bubbles were treated as sinks. Calculations were carriedout with respect to the change in gas concentration as a function oftime using arbitrary units. Gas concentration is indicated on a colorscale with red 90 being high and blue 92 being none. Bubbles 94 on theright side of the plot are spaced more closely than the bubbles 96 onthe left axis, which are spaced farther apart. After a fixed diffusiontime, gas dissolved in the polymer is fully depleted between the closelyspaced features 94, suggesting bubble growth would be limited, whereasgas is still available between the larger spaced bubbles 96. Factorsthat affect the microbubble formation include, but are not limited to,polymer thickness, gas concentration, cure temperature, cure time, andpit depth.

In a method of using the microbubble arrays, fluid flow properties areexamined. An example of a use of the arrays is for the deposition ofcancer cells using flow or gravity to sustain these cells in amicroculture. FIG. 11 shows a spherical cavity 100 made in accordancewith this embodiment of the invention disposed in a substrate 102 havingcultured cells 104 therein.

A key requirement for utilizing microbubble arrays in cell sorting andmicrocell culture, examples of two forms of use of the microbubble arrayherein, is the ability to selectively and spatially alter themicrobubble surface chemistry. FIG. 12 is a schematic diagram of avacuum-assisted coating (VAC) procedure 110 used to selectively coatbioactive molecules onto a microbubble wall 112 and the microchannelsurface 114. The capability is desirable as it may be advantageous tocoat the microchannel surface 114 with a blocking agent, such as bovineserum albumin (BSA) or polyethylene glycol (PEG) to prevent nonspecificcell interactions while depositing a chemotactic (e.g., stromal-derivedfactor-1 (SDF-1), IL-8) or extracellular matrix molecule, such as aprotein (e.g., collagen, fibronectin), or an adhesive ligand or receptor(e.g., selectin, antigen, antibody) in the microbubble wells 112 toenhance cell capture, adhesion or bioactive molecule to direct cellfate. The VAC process takes advantage of and is used to overcome theintrinsic PDMS polymer hydrophobicity (θ˜105°).

As an example, the VAC process may begin with Step (a), where thepolymer sample is exposed to UV-ozone (BioForce Nanosciences ProCleaner)for 1 hour. Although used in this non-limiting example, the VAC processmay be conducted without the use of this ozone step. Alternative methodsmay also be used to render the surface hydrophilic including, but notlimited to, the application of oxygen gas plasma. This treatment makesthe ozone-contacted surfaces hydrophilic (θ<105°). A shadowing effectoccurs that renders the undersurface at the microbubble entrance 12sufficiently hydrophobic to inhibit aqueous solutions, gently dispensedon the planar surface 14, from entering the microbubble 12, as shown inStep (b). The application of a vacuum in Step (c) indicates that it ispossible to coat bioactive molecules on the microchannel surface 14 thatmay or may not differ from those deposited in the well 12.

Microfluidic flows using microparticle image velocimetery of isolatedmicrobubbles (nearest microbubble is greater than about 10× bubblediameter opening) with square (80 microns) opening reveal asymmetricfluid flows above the entrance that extend into the cavity. Asymmetricflows depend on microbubble size and fluid shear stress. Under aconstant shear stress of 5 dyn/cm² (about 0.2 ml/min) microparticlesenter the cavity at the downstream edge. Stable asymmetric recirculatingvortices develop causing the microparticles to exhibit a triangularvelocity trace in the cavity with fore-aft asymmetry. Along themicrobubble well bottom they flow counter to the main stream. At the topof the cavity, they flow parallel to the main stream. This flow/shearstress profile can be altered by changing the flow velocity and/ormicrobubble geometry. This enables one to customize the mixing andnutrient/waste exchange in LOC applications.

FIG. 13 is just one example of a device 120 that incorporates themicrobubble array of the embodiments of the present invention. Asubstrate 122 includes an optical biosensor 124 such as Porous Silicon(PSi) embedded therein. A spherical cavity 126 is disposed in substrate122. A chemotractant, biomolecular ligand, and flow properties causestarget cells to form the cell mixture 128 into the spherical cavity 126.The PSi optical sensor 124 is designed to detect certain types of cellsor cell products, collected in spherical cavity 126, or to monitorspecific chemical signals (cytokines, gases, small molecules) or cellconcentrations within spherical cavity 126 in real time.

As described herein, a microfluidic device is provided using GEM moldingand VAC techniques. The microbubble features are applicable todiagnostic, therapeutic, and investigative purposes in the many researchareas including rare cell sorting, high throughput screening, and stemand cancer cell research. Examples of devices that can incorporate themicrobubble feature include, but are not limited to, devices for drugscreening, pharmacokinetic analysis, cytotoxicity analysis, cellcapture, quorum sensing, isolation of cells, and chemotherapeuticresponse of cells.

EXAMPLES Example 1 Effect of Wafer Coating on Microbubble Formation

The PDMS pre-polymer is applied to the silicon wafer mold as describedin the Materials and Methods. Prior to curing, the viscous pre-polymerdoes not readily infiltrate the pits of the wafer mold due to thesurface tension between the pre-polymer and the hydrophobic moldcoating. This allows for meniscus nucleation and microbubble growthabove the mold opening during curing. Three types of hydrophobiccoatings, the Bosch process passivation coating, PTFE plasma depositedcoating, and the applied FDTS coating were examined to determine themicrobubble formation efficiency (MBFE=the # of microbubbles thatform/total # of mold openings). These coatings had measured watercontact angles (θw) of ˜105°, ˜120°, and ˜105°, respectively. Each ofthe three hydrophobic coatings were tested by molding PDMS onto apatterned silicon wafer containing 10×10 arrays with circular and squaremold openings 100 μm in diameter and depth of 100 μm. The Bosch processpassivation layer had a MBFE of >99% for both 100 μm square and circleopenings in 10×10 arrays indicating the most consistent formation ofmicrobubbles. It was also observed that the Bosch coating performed bestwith pit openings of other sizes ranging between 60-200 microns.Interestingly, for square openings the plasma deposited PTFE coatingperformed the worst. Reasons for this are uncertain but likely are dueto inefficient PTFE deposition (0.2 μm) over mold features with sharpcorners. MBFE may be improved with increasing the thickness of the PTFEcoating. The higher MBFE achieved with the FDTS coating applied byliquid immersion is likely due to the fact that the solution infiltratesthe mold features to deposit a more uniform coating in and around thesquare pit.

Example 2 Effect of Wafer Mold Opening Size and Shape

The silicon wafer mold opening size and shape both influence thedimensions of the microbubbles that form above them. To quantify these,analysis includes only the 6×6 matrix of the inner microbubbles of a10×10 array, due to spatial edge effects discussed below (FIG. 14,inset). Results show that increasing the diameter of the mold openingleads to a linear increase in microbubble diameter for a PDMS chipthickness of ˜1 mm, as shown in FIG. 14 (where the pit depth is 100 μm,and showing the diameters of the inner microbubbles of a 10×10 array(inset) for circle (∘) and square (▪) opening diameters of 60, 80, 90and 100 μm (n=72)). Two representative sets of 6×6 inner arrays, moldedin the same PDMS cast, were analyzed (n=72 microbubbles) for each sizedopening. The correlation coefficient R² for the square and circleopenings is 0.99 and 0.92, respectively. Similar uniformity trends areobserved in subsequent PDMS casts from the same silicon mold. Thepositive correlation between microbubble size and mold opening isrelated to the microbubble formation mechanism. The mold openingnucleates the formation of a meniscus in the PDMS pre-polymer due toexpansion of gas in the pit during the rapid heat cure process. Residualgas in the PDMS pre-polymer diffuses to the meniscus and fuses to formthe microbubble compartment. Microbubble growth ceases when residual gasis depleted (as shown below) and/or when the PDMS cures. Increasing thesize of the mold opening increases the surface area of the nucleationsite allowing for larger microbubbles to form.

The shape of the pit opening also contributes to the dimension of themicrobubble. Circular openings form microbubbles that are statisticallylarger (p<0.0001) than those formed over square openings for pit openingdimensions ranging between 60-90 μm diameter (see FIG. 14). Reasons forthis possibly relate to the presence of stress at the pit opening. Forthe circular geometry, forces distribute evenly along the periphery ofthe mold opening, allowing for more even microbubble growth. Conversely,cohesive forces exerted at the sharp corners of the square opening mayhinder microbubble expansion. This phenomenon likely contributes to thelower MBFE observed for square openings. A similar finding was reportedin a recent study of microbubbles generated by electrolysis, which foundmicro-fabricated circular electrodes formed bubbles more reproduciblyand with more uniform size compared to square or triangular electrodes.However, it was also observed that when the mold opening is 100 μm indiameter the square openings produce statistically larger diametermicrobubbles, 117±3.2 μm, compared to those formed over circularopenings, 112±1.8 μm (p<0.0001). This transition is likely due to subtledifferences in microbubble shape which lead to constriction in bubblegrowth. Cross-sectional analysis reveals that microbubbles formed overcircular pits adopt a slight obtuse shape in contrast to the nearperfect spherical cross-section formed over square mold openings. It ispossible that microbubble expansion over large closely spaced circularmold features becomes laterally constricted. The nonobvious phenomenonof spatial constraint is discussed further below.

Example 3 Effects of Spatial Arrangement of Wafer Mold Openings

Parametric studies on microbubble formation in PDMS have shown thatthere are nearest neighbor spatial effects on the size of themicrobubbles that form in an array format (see FIG. 14, inset).Microbubbles formed at the corners of the 10×10 array are larger thanthe edge row microbubbles, which are larger than interior microbubbles.The relative size of microbubbles in an array depend on the specificspatial arrangement of pit openings. For example, microbubbles at thecorners of a 10×10 array range between 1.5 to 2.6 times larger indiameter than (constricted) microbubbles in the array interior. Thisphenomenon can be attributed to a competition for available gas trappedin the PDMS prior to the cure point which dependes on PDMS thickness.Microbubbles that are unconstricted have more gas available to them andface less competition from adjacent nucleation sites. Thus, cornermicrobubbles grow larger than edge row, which are larger than interiormicrobubbles which are the most constricted. Comsol simulation studiesof gas diffusion from the bulk to microbubbles supports this idea (seeFIGS. 10 and 15A, where color coding indicates high (red) to low (blue)gas concentration at steady state). Microbubbles sinks were spaced anincreasing distance apart and when steady state diffusion conditionswere attained, results show that the gas concentration is significantlydepeleted between constricted (closely spaced) microbubbles. A highergas concentration is evident between microbubbles spaced far apart(unconstricted). This phenomenon also explains why increasing the PDMSthickness produces larger microbubbles. Thicker PDMS films providelarger dissolved gas reserviors.

Example 4 Effect of Mold Feature Depth and Uniformity of MicrobubbleFormation

The effect of mold feature depth, ranging from 25 μm to 100 μm, onmicrobubble formation was also investigated. The deeper the featuredepth the more air that is trapped when the PDMS is poured onto thewafer mold. Results reveal, however, that similarly sized microbubblesform independently of depth. For all feature depths, the volume of themicrobubble formed far exceeds the volume of air in the featuretheoretically expanded at 100° C. This provides further support for theimportance of dissolved air in the PDMS pre-polymer contributing tomicrobubble formation. Mold feature depth is observed, however, to be animportant factor in contributing to the reproduciblity of microbubbleformation sample-to-sample (i.e. repeated PDMS molding from the sameDRIE wafer). Pit depths >50 μm are required for repeated use of wafermolds to form microbubbles with high MBFE cast-to-cast. This isattributed to the thickness of the hydrophobic coating that forms withlonger DRIE etch times. For example, with 100 μm deep features it ispossible to mold unconstricted microbubbles over 60, 100 and 200 μmdiameter openings with consistent size across a 4-inch wafer. Detailedanalysis showed efficient microbubble formation MBFE >98% withmicrobubble diameters that varied <10% across the 4-inch chip. Tightercontrol of PDMS thickness would increase the uniformity of microbubblediameter across large areas. Locally within a 10×10 array (see FIG. 14)the microbubble diameter coefficient of variance was <1%. These datademonstrate that uniform, homogenously sized microbubble arrays can befabricated over a large area (˜6 in²). Moreover, provided that thehydrophobic coating on the mold is not damaged mechanically or bycleaning with inappropriate solvents, a mold can be repeatedly used withno known limit. This makes microbubbles a cost advantaged technology.The fact that microbubble arrays can be used as a mold to cast newarrays makes this technology extremely affordable.

Example 5 Effects of Cure Time at 100° C. on Microbubble Formation

Studies suggest that there is a dependence of microbubble formation onPDMS curing time at 100° C. cure temperature. PDMS can be cured at roomtemperature, but this does not result in microbubble formation. Curingat a temperature >65° C. is required to form microbubbles. Hightemperature is needed to expand gas trapped in the pit and to facilitatediffusion of gas dissolved in the polymer premix to feed microbubblegrowth at nucleation sites above each pit. Studies were conducted toinvestigate the kinetics of microbubble formation. Although the standardprocedure is to cure PDMS for 2 hr at 100° C., results shown in FIGS. 23and 24 indicate that microbubbles form within the first 5 minutes ofcuring at 100° C. In these studies a sample, comprised of polymer premixcast onto a silicon mold with etched pits in 10×10 array, is placed inthe 100° C. oven for a fixed time ranging from 1 to 90 minutes. Afterthe fixed time the sample is removed from the oven and allowed to finishcuring at room temperature. Microbubble size measurements were madeafter 24 hr on the inner 6×6 array only (n=72). Microbubble formation isevident following just 1 minute in the 100° C. oven however, microbubbleformation efficiency is low and microbubble size in not uniform acrossthe array. Curing the sample for ≦10 min at 100° C. is sufficient toform microbubble with uniform size across the array.

Example 6 Effects of PDMS Thickness on Microbubble Size

It was possible that there is a dependence of microbubble size on thethickness of the polymer layer. In general, the volume of themicrobubbles that form far exceeds the volume of gas trapped in the moldpit expanded at 100° C. cure temperature. This observation supports thekey role that dissolved gas in the polymer has in diffusing to nucleatedbubbles supporting microbubble growth. The thicker the polymer thelarger the dissolved gas reservior. An example set of data illustratingthis effect and the ability to mold homogenously size microbubbles overlarge arrays is given in FIGS. 25A and 25B. FIG. 25A illustrates regions(inner, middle, and outer) on a 4 inch diameter silicon mold that chipswere fabricated with constrained microbubble (pit spacing 3× openingdiameter) and used for size measurements. FIG. 25B summarizesmeasurements of microbubble maximum diameter as a function of PDMSthickness ranging from 0.5 mm to 4 mm. All microbubbles hadapproximately 100 μm diameter circular openings in these particularexperiments. Microbubble diameters range from ˜100 μm with PDMSthickness of 0.5 mm (aspect ratio ˜1) to ˜160 μm with PDMSthickness >2.5 mm (aspect ratio ˜1.6). Microbubble size asymptotes above2.5 mm suggesting the maximum size is constrained by nearest neighboreffects. Increasing the spacing between pits on the mold will allowformation of larger microbubbles.

Example 7 Effects of Curing Temperature of PDMS Pre-Polymer

Studies suggest that there is a dependence of PDMS curing temperature onformation and the size of microbubble. FIG. 15B shows the diameter ofthe constricted (▴) and unconstricted (▪) microbubble and their MBFE (∘and ×, respectively) as a function of curing temperature (n=20 and errorbars are standard error). For curing temperatures ≦40° C. microbubblesform inconsistently with different sizes causing large standard error.At curing temperatures ≧65° C. microbubbles form with near 100%efficiency and their sizes are nearly temperature independent. This datasuggests that temperatures ≧65° C. are needed to attain the gasexpansion and diffusion requirements for microbubble formation. At lowertemperatures, there is insufficient free energy available tomechanically and thermodynamically overcome the interfacial tensionbetween the viscous PDMS pre-polymer and the air trapped in the moldfeature and/or gas diffusion through viscous premix is slow which favorsreverse molding or inconsistent microbubble formation.

Example 8 Effect of Residual Gases in the PDMS Pre-Polymer

Microbubbles are unable to form in PDMS which has been degassed. Thisresult further supports the theory that the formation of the microbubbleis, in large part, due to the recruitment of gases in the pre-polymer tothe nucleation site at the openings in the silicon wafer mold. In orderto investigate the effects of the composition of residual gas in thepre-polymer on microbubble size, a series of degas/regas experimentswere conducted. First, the PDMS pre-polymer was degassed in vacuum (−690mmHg relative to atmospheric pressure) for one hour and subsequentlyaerated with different gases (N₂, Ar, CO₂, He, and compressed air)before curing. Microbubble size was then determined and tabulated.Results show that the size of the microbubble and the MBFE compared tonon-degas sed controls depends on the composition of the gas aeratedinto the PDMS pre-polymer.

FIG. 16A shows the average normalized microbubble diameter formed fromaerated samples as a function of regas time compared to controls. Thegeneral trend observed is that CO₂ forms the largest microbubbles,achieving between 60-100% recovery of the control microbubble diameter.Nitrogen also formed large microbubbles ranging between 60-90% ofcontrol. The other three gases (Ar, He, and compressed air) formedmicrobubbles between 40-75% of the control diameter. These trends areconsistent with expectations based gas solubility (S), and the rate ofdiffusion (D) of these gases in PDMS. Gas permeability (P) is given bythe product of S*D. As shown by others, CO₂ has the highest solublitythrough PDMS (400×10² cm³(STP)/cm³ atm) and He has the lowest (0.03×10²cm³(STP)/cm³ atm). This suggests that after aeration with CO₂, a higherconcentration is sustained in the viscous PDMS pre-polymer compared tothe other gases. Similarly, N₂ has a high solubility (110×10²cm³(STP)/cm³ atm, and is expected to form large microbubbles, butsmaller than CO₂ as is observed. In contrast He, due its inertness andsmall size, has a low solubility and higher D (5.6×10⁸ cm²/s) comparedto N₂ (1.3×10⁸ cm²/s). Hence, aeration with He forms microbubbles onaverage only half the size of control. These results confirm that abalance is necessary among the physical properties of the gases in thePDMS that influences the resulting microbubble size.

The corresponding MBFE is also dependent on the type of gas that isaerated into the degassed PDMS pre-polymer (see FIGS. 16A and 16B).Aeration with nitrogen, argon, and compressed air samples formedmicrobubbles most efficiently (MBFE >80%), as shown in FIG. 16B. Whilecarbon dioxide recovers microbubble size to the greatest degree, itforms them inconsistently (MBFE ˜50%). Nitrogen is the only gas tohave >80% recovery of microbubble size compared to native air and MBFE˜80%. Since nitrogen is the major component of ambient air, thissuggests that its presence may facilitate consistent microbubbleformation. Further studies would be necessary to optimize the aerationprocedure to be able to control microbubble size and formationefficiency by this technique. Nonetheless, these results prove thatdissolved gas in the pre-polymer is required for microbubble formation.

Example 9 Advantages of Microbubbles for Cell Culture

One goal is to advance microbubble technology for cancer and cellbiology research and to demonstrate the unique advantages of thisarchitecture over existing technologies; namely standard tissue culturepolystyrene (TCP) and microfabricated rectilinear wells. A typicalmicrobubble with a 100 μm opening and a 250 μm diameter has a volume of8.18 nL. This is more than 4× larger than the volume of a cylindricalrectilinear well with the same sized opening and depth (1.96 nL). Theincreased microbubble volume minimizes the susceptibility to verticaldiffusion limitations which is commonly observed in rectilinear wellswith aspect ratio (>1). Plating 10 cells per microbubble (250 μmdiameter) has a near equivalent cell plating density of ˜5000 cells/cm²in a standard 96-well TCP (0.32 cm²) seeded with ˜1600 cells per well.However, the microbubble inherently provides a ˜75× lower volume ofmedia per cell compared to a 96 well plate (100 μL) assuming minimalfluid exchange with a media reservoir external to the microbubble. Thisallows for the concentration of secreted factors produced by cells torise to high levels quickly, creating a local microenvironmental nichethat can influence cell function. In the following, three applicationsof microbubble technology are presented that exemplify this and otheradvantages for cell culture. Examples include 1) single cell culture inmicrobubbles, 2) formation of arrays of homogenously sized 3D cellaggregates, and 3) demonstration through experimentation and simulationthat the bubbular architecture provides a microenvironmental niche thatcan influence cell response. These studies confirm that cells culturedin microbubbles can condition their microenvironment, enabling theirsurvival and proliferation to adopt a characteristic cell colonyphenotype. These examples demonstrate that microbubbles constitute anovel in vitro tool that can transform basic studies in cell biology,cancer and stem cell research.

Example 10 Microbubbles Support Single Cell Culture

The compartmentalized architecture of the microbubbles and the abilityto alter the PDMS surface chemistry are features that can be exploitedin conducting single cell culture studies. A single YUSIK (metastaticmelanoma) and a single HaCaT (non-tumorigenic keratinocyte) cell werecultured in plasma-treated hydrophilic and untreated hydrophobicmicrobubbles. Both cell types exhibit spreading and proliferation inplasma-treated microbubbles at 24 and 72 hours, respectively. Incontrast, single cells seeded in untreated hydrophobic microbubbles donot spread or proliferate even after 120 hours. Results for a singleHaCaT cell are shown in FIGS. 17A and 17B. Presumably, without cell-cellcontacts, an ˜8.1 nL microbubble is too large for a single cell tocondition its environment to enable its adhesion and proliferation in ahydrophobic well. It will be shown below that increasing the number ofcells in a similarly sized hydrophobic microbubble does promote cellsurvival and proliferation. It is interesting to note the strong effectsurface energy has on cell adhesion and proliferation. This resultdemonstrates that hydrophilic microbubbles can be used to supportproliferation of a single cell. Single cell survial in hydrophobic orhydrophilic microbubble is dependent to a large extent on cell type.This culture system can be used to identigy tumor initiating cells orcancer stem cells, as these cells have a priviledged capacity tocondition their microenviorment in an autocrine fashion. The microbubblesurface may further coated with bioactive substance to enable singlecell surivial. Using a vacuum-assisted coating technique, microbubblesurfaces can be treated with a wide range of bioactive proteins tofurther alter and investigate single or multiple cell response.

Example 11 Forming Arrays of Homogeneously Sized 3D Cell Aggregates

Cell-cell contacts and cell-substrate interactions are importantbiological stimuli that influence the survival and structuralorganization of cells. Cell adhesion to the extracellular matrix is akey factor in tissue homeostasis. Anchorage-independent cellproliferation is a recognized hallmark in metastases and tumorigenesis.It was observed that in vitro some cancer cells can proliferate onhydrophobic surfaces and adopt a colony morphology that differs fromstandard TCP. This is exemplified by YUSIK metastatic melanoma cells asshown in FIGS. 18-20. As shown in FIG. 18, on TCP YUSIK cells grow in amonolayer whereas on planar hydrophobic PDMS they do not spread into amonolayer but proliferate rapidly in 3D aggregates. Results from an MTTassay, depicted in FIG. 19, shows YUSIK cells are more active on PDMSthan on TCP. Although the 3D morphology may be a more physiologicalrelevant model for biological or chemotherapeutic studies; it ishowever, difficult to control the size of the aggregates on planarsubstrates. Microbubbles provide a simple means to confine the growth ofhomogeneously sized 3D cell aggregates in an array format. YUSIK cellswere seeded (˜10 cells per microbubble) in untreated hydrophobicmicrobubble wells and cultured at 37° C. and 5% CO₂ for 9 days. Theclustered phenotype was adopted within 24 h of cell seeding and by day6, YUSIK cells were observed to completely fill the wells. Multiphotonmicroscopy images of samples stained with calcein-AM (live, green) andpropidium iodide (dead, red) show that cells throughout the filledmicrobubbles were viable (see FIG. 20). No evidence for necrotic cellswas observed in these experiments. Cultures can be sustained for 14 daysor longer, suggesting that microbubble cell culture is not limited bynutrient/waste exchange through the microbubble opening despite the ˜250μm diameter size of the microbubble compartment. Rectilinear microwellscould in principle be used to confine the growth of 3D cell aggregates,but due to vertical diffusion gradients shallow wells with aspect ratio(depth/width) <1 would be needed and this would not allow foraccumulation of soluble factors to influence colony phenotype as isdemonstrated below.

Example 12 Cellular Response to Conditioned Microbubble Environment

For in vitro cell culture, the media volume to cell ratio is animportant factor driving cell survival and proliferation. Cells rely onsecretion of soluble factors (ie. growth factors, cytokines) toencourage adhesion and proliferation, particularly on uncoatedsubstrates where cells must synthesize their own matrix or matrix isabsorbed from media. When cells are seeded onto unfavorable substratesthey may migrate to form cell-cell contacts to survival. The ability ofthe screted factor concentration to rise to bioactive levels depends onthe media volume to cell ratio and factor production rate. Here it isshown through simulation and experiments that microbubbles offersignificant advantages over existing technologies to enable cells tocondition and respond to their microenvironment.

Two dimensional mass transport simulations were conducted to simulatethe diffusion of soluble factor secreted by a 10 μm cell placed at thebottom of both rectilinear and microbubble wells with varying aspectratio. At equilibrium the concentration profiles at the well openings,vertically through the wells, and the integrated concentration in thewells were examined. Results, depicted in FIG. 21, show a general trendthat for rectilinear wells the vertical concentration profiles arelinear. In contrast, the architecture of the microbubble and itsincreased volume produce a varying concentration profile that depends onthe aspect ratio. Shallow wells (aspect ratio <1) leak secteted factorout of the well and it is diluted in the bulk media reservior. Foraspect ratios >1, the concentration at the microbubble openings arehigher than for rectilinear wells. Microbubbles show an advantage inallowing the secreted factor concentration to rise to higher levels anddistribute more evenly throughout the microbubble at high aspect ratio.This is due to the increased volume which allows for development of amore shallow gradient and accumulation of soluble factor. Thesesimulations support the notion that soluble factor can accumulate inmicrobubble wells and may be less prone to suffer from verticaldiffusion limitations known to occur in high aspect ratio rectilinearwells. For this reason, rectilinary microwells are typically fabricatedfor cell culture with aspect ratio <1. The simulations show that forshallow rectilinear and hemispherical wells (aspect ratio <1) solublefactor is readily lost to the external media reservoir (see FIG. 21).

To provide experimental evidence that soluble factor secreted from cellsseeded in microbubbles influence their response, studies were conductedof HaCaT cells seeded at near equivalent density (˜1×10⁴ cells/cm²) inhydrophobic microbubble and on hydrophobic planar PDMS cast in 96-wellTCP. Results show a fascinating change in HaCaT morphology in themicrobubble that does not occur for cells culture on planar PDMS. Onplanar PDMS the cells migrate to form a single compact spheroid observedat ˜24 h (see FIG. 22A). Due to the curvature of the microbubble HaCaTcells settle in a cluster at the bottom and in 24 h also form a singlecompact spheroid as shown in FIG. 22B. Interestingly, by 48 h HaCaTcells are observed to undergo a morphological transformation in themicrobubble to adopt a sheet morphology. On planar PDMS this transitiondoes not occur even after culturing for 6 days with or without mediachanges. The ability of HaCaT cells to undergo this morphology change isattributed to accumulation of soluble factor secreted by cells tobioactive levels in the microbubble. HaCaT spheroids are unable tocondition their media in the 96-well format. By day 6, cells on planarPDMS begin to die and the spheroid dissociates. Studies are underway toinvestigate biochemical differences between the spread and clusteredcolony phenotypes, to identify the soluble factor, and to determine thedependence of this morphology transition on microbubble volume and thenumber of cells seeded in a microbubble.

Materials and Methods

Molding Microbubbles in PDMS Using a DRIE Silicon Wafer

To characterize the parameters that impact microbubble size a siliconmold using AutoCAD LT 2008 (Autodesk, Inc., USA) was designed that hadmold openings arranged in three different spatially oriented arraysincluding 10×10, staggered, and alternating rows with opening shapesincluding circles, squares, triangles and rectangles. The diameters ofthe mold openings ranged from 20 to 200 μm. Five silicon wafer moldswere produced from this mask (MEMS and Nanotechnology Exchange, USA).Three wafers were etched at different feature depths (25, 50 and 100 μm)using the Bosch deep reactive ion etch (“DRIE”) process (Plasma Therm770). These wafers were received following the Bosch DRIE process withthe resist and hydrophobic coating intact. Using a Ramé-Hart Goniometer,a water contact angle (Θ_(w)) of ˜105° was measured. The remaining twowafers had etched depths of 50 μm. For these, the resist and hydrophobiccoating were removed using a photoresist stripping process (Metroline)that rendered the wafer surface hydrophilic. One hydrophilic wafer(Θ_(w)<20°) was received directly and the other was received followingplasma deposition of a 0.2 μm polytetrafluoroethylene (PTFE) film(Θ_(w)˜120°).

Hydrophobic FDTS Coating Technique

A mixture of perfluorododecyl-1H,1H,2H,2H-triethoxy-silaneperfluorotetradecyl 1H,1H,2H,2H-triethoxy-silane (FDTS; GelestSIP6720.5) was diluted to a 2 mM solution in heptane. The receivedhydrophilic DRIE wafer was rinsed with ethanol and dried under N₂ gas.The wafer was submerged into 20 mL of the prepared FDTS solution in aglass Petri dish and placed on a rocker for 15 min. The coated wafer wasremoved from the solution and immediately dried under N₂ gas. Excesssolvent was further removed by incubating the wafer at 100° C. for 30min. This coating had a measured water contact angle (Θ_(w)) of ˜105°.The FDTS coated wafer was subsequently used as a mold for PDMS curing asdescribed below.

PDMS Curing

The DRIE silicon wafers were used to mold PDMS microbubble chips withDow Corning's Sylgard® 184 silicone elastomer kit in a 10:1 base tocuring agent ratio (w/w). This pre-polymer was manually mixed with apipette tip in a 50 mL conical tube for 30 seconds and then poured ontothe silicon wafer molds. The mixture was allowed to self-level for 30minutes at room temperature, and then cured at 100° C. for 2 h, exceptwhere noted for specific experiments. For studies performed inPDMS-coated 96-well plates, 50 μL of the PDMS pre-polymer was pipettedinto each well and allowed to settle at room temperature for 30 min. Theplates were then cured at 40° C. for 4 hours. This lower curingtemperature was used to ensure that the tissue culture plate remainedunchanged by the curing process.

PDMS Aeration Studies

Previous work suggested that the residual gases in the PDMS pre-polymerare essential for microbubble formation. Here, an in-depth investigationof the influence of the residual gas composition by aeration experimentsusing nitrogen, argon, carbon dioxide, helium pure gases (Air Gas, Inc.,USA), and compressed air is provided. Excluding compressed air, thesegases are known to make up a significant portion of the Earth'satmosphere: 78.1%, 0.9%, 0.04%, and 0.0005%, respectively. In thismethod, the PDMS pre-polymer was poured into a weigh boat and degassedin a bench-top vacuum chamber at −690 mmHg relative to atmosphericpressure, for 30 min to remove the residual gas from the pre-polymer.Applying the degas sed pre-polymer to the hydrophobic DRIE mold yieldedthe expected reverse mold. After degassing, the mixture was pipettedinto 2 mL Eppendorf tubes and aerated with a gas at a pressure of 30 psifor 0, 0.5, 1, 2, 5, or 10 min. Then, the aerated samples were pouredonto a wafer mold, allowed to self-level at room temperature for 30 min,and cured at 65° C. for 3 hours. It was shown (FIG. 15) that thisreduced curing temperature does not significantly affect the diameter ofthe microbubble or the microbubble formation efficiency, defined below.PDMS film thickness was 1±0.1 mm as film thickness impacts microbubblesize (see, e.g., FIGS. 25A and 25B). Increasing PDMS thickness produceslarger bubbles with all other mold conditions kept constant.

Comsol Simulations of Gas Diffusion in Microbubbles

Two dimensional mass transport simulations were conducted using theChemical Engineering Module of Comsol Multiphysics (Comsol, USA) toinvestigate (1) the effect of microbubble spacing on the diffusion ofgases to microbubble structures and (2) the effect of the well aspectratio on the diffusion of soluble factors produced by a cell cultured inthe well. The Comsol Multiphysics modeling software was used to simulategas diffusion from the bulk to 8 microbubbles with 100 micron openingsspaced an increasing distance apart. Microbubble surfaces were set assinks (c=0 mol/m³) and all other surfaces were set as insulatorboundaries (no concentration exchange). The bulk fluid was set at aninitial concentration of c=1 mol/m³. Steady state results showed theconcentration of soluble gases around the microbubbles.

The diffusion of soluble factor secreted by a 10 μm cell (source) placedat the bottom of a well was also simulated. Specifically, differences inthe concentration profiles between microbubbles and rectilinear wellswith aspect ratios varying from 0.5 to 20 were investigated. The initialboundary conditions were set at the cell source (c=1×10⁻⁶ mol/m³ or 1nM) and the walls (c=0 mol/m³). For the simulations, the free diffusioncoefficient of epidermal growth factor (EGF) in dilute agarose(D=16.6×10⁻⁷ cm²/s), which was determined by others, was used. Theequations were solved to determine the concentration as a function oftime until steady state was reached.

Analysis of Microbubble Formation in PDMS

Parametric studies were carried out on cured PDMS films cast onto eachwafer from MEMS Exchange. Microbubble arrays were imaged using phasecontrast microscopy (Olympus IX70 with QImaging Retiga EXL camera).Images were processed using ImageJ software (NIH, USA). Measurements ofmicrobubble diameter were made from top-view images and results werecompared to the cross-sectional diameter obtained by using a razor tocut the microbubble chip. The diameters measured by both methods werenot statistically different (p>0.05).

Cell Culture Using PDMS Microbubble Technology

A metastatic melanoma cell line (YUSIK) and an immortalized keratinocytecell line (HaCaT) were used to demonstrate advantages of microbubblesfor cell culture. These cells were grown at 37° C. with 5% CO₂ inOpti-MEM reduced serum medium (Gibco 31985-070; Invitrogen Corp., USA)and DMEM (Gibco 11995-065, Invitrogen Corp., USA), respectively, andboth were supplemented with 5% heat inactivated fetal bovine serum(Gibco 10082-147, Invitrogen Corp., USA) and 1% Penicillin/Streptomycin(Gibco 15140-122, Invitrogen Corp., USA). To prepare microbubble chipsfor cell culture, they were first soaked in ethanol solution for 10seconds in a sterile hood and blown dry with nitrogen. The bottom of themicrobubble chip was rendered hydrophilic by a 30 min. exposure toUV-ozone using a UV/Ozone ProCleaner (BioForce Nanoscience, Inc., USA).This was done to keep the sample submerged in the media after seedingthe cells. For experiments involving single cell culture only, anadditional step was done in which the top of the microbubble chip wastreated in gas plasma (March Plasmod) for 10 min. at 20 W to rendersurfaces hydrophilic. Then, the top surface was blocked with 50 μL of 1%bovine serum albumin (Hyclone SH30574.01, Thermo Scientific, USA) for 30min.

To fill microbubble wells with cell culture media a vacuum-assistedcoating (VAC) technique previously described was utilized. Briefly, PBSbuffer (40 μL) was pipetted onto the top of a sterile 1.0×0.5 cmmicrobubble chip. It was then placed in a desktop vacuum chamber.Application of negative pressure (−690 mm Hg relative to atmosphericpressure) for 30 min depletes gases trapped in the microbubbles, causinga rapid injection of solution into the microbubble well. The primedmicrobubble compartment allows for its further use in cell culturedescribed below.

A reagent exchange process was used to fill the microbubble with cellculture media. Here, the PBS buffer was removed and 50 μL of cellculture media was applied to the buffer primed microbubble chip surface.The buffer solution inside the microbubble mixes with the applied cellmedia. Pipette mixing facilitates the exchange. This process is repeateda second time to pre-condition the microbubble for subsequent cellseeding. Next, 50 μL of cell stock solution (2×10⁵ cells/mL) was appliedto the microbubble chip for 15 minutes at a cell density of 2×10⁴cells/cm². Studies show that this process seeds on average 10 to 20cells per microbubble. For single cell studies that are described below,cells were incubated for 1 min. at a cell density of 4×10⁴ cells/cm².After seeding, the cell solution was removed from the chip by pipetteand the chip was rinsed twice with 50 μL of new media. This step wasdone to gently remove cells that had deposited onto the PDMS surface.Next, the chip was transferred with sterile forceps to a new well in a24-well plate pre-filled with 1 mL media and the plate was placed in anincubator at 37° C. and 5% CO₂. Culture media was exchanged every threeto four days. Cells were cultured for over 14 days. A viability assaywas performed using calcein-AM (live, green) and propidium iodide (dead,red) used at 1.0 μM and 1.5 μM, respectively. Fluorescence images weretaken using a multiphoton microscope (Olympus Fluo-view FV 1000 AOM-MPM)with band pass filters 519/26 nm (OPI#08) and 565/40 nm (OPI#11) forcalcein AM and propidium iodide, respectively.

Cell Proliferation Assay

To determine the extent of proliferation of cells grown on planar,hydrophobic PDMS surfaces compared to standard TCP, cells were seededonto PDMS cured 96-well TCP. A colorimetric MTT assay,3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), was usedto detect mitochondrial enzyme activity using a Modulus II MultimodeMicroplate Reader (Turner BioSystems). The yellow MTT dye is reduced bymitochondrial reductase enzymes to form purple formazan crystals thatcan be dissolved and quantified by absorbance measurement which providesan indirect measure of cell viability based on metabolic activity. Thistest could not be carried out for cells cultured in the microbubblebecause the absorbance of the dye produced by the low concentration ofcells is undetectable by currently available means. Proliferation inmicrobubbles can be indirectly inferred by the time it takes for cellsto migrate out of the well onto the planar chip surface.

While the invention has been described with reference to exemplaryembodiments, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing from theessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments falling within the scope of the appendedembodiments.

What is claimed is:
 1. A method of manufacturing a microfluidic devicecomprising: providing a mold having one or more pits therein; applying aan uncured polymer liquid onto the mold, covering the one or more pitsto create an interface between the uncured polymer liquid and the mold;allowing the mold with said uncured polymer liquid thereon to sit curingthe uncured polymer at an elevated temperature to form a cured polymer,wherein gas at the liquid mold interface expands during curing to formone or more curvilinear cavities in the cured polymer, wherein each ofsaid curvilinear cavities comprises an inner surface and an opening tothe exterior, the opening having a first diameter, wherein the innersurface of each curvilinear cavity curves outward from a rounded bottomlocated at a point furthest from said opening of said cavity to amaximum diameter, and then curves inward from said maximum diameter tosaid opening, said maximum diameter being greater than said firstdiameter: and separating the cured polymer from the mold to provide adevice in the form of a polymer substrate having one or more curvilinearcavities therein.
 2. The method of claim 1, wherein the ratio of themaximum diameter to the first diameter is greater than
 1. 3. The methodof claim 1, wherein the maximum diameter is approximately 50 to 350microns.
 4. The method of claim 1, wherein the first diameter isapproximately 60 to 200 microns.
 5. The method of claim 1, wherein thecurvilinear cavities are spherical, obtuse, or oval in shape.
 6. Themethod of claim 1 wherein the step of allowing the mold with saiduncured polymer liquid thereon to sit further comprises allowing gastrapped in the one or more pits to combine with gas diffusing from theliquid.
 7. The method of claim 1 wherein the step of allowing the moldwith said polymer liquid thereon to sit is conducted for about 10 toabout 60 minutes at a temperature in the range of about 60 to about 200°C.
 8. The method of claim 7 wherein the temperature is in the range ofabout 50 to about 100° C.
 9. The method of claim 1 wherein the step ofallowing the mold with said uncured polymer liquid thereon to sit isconducted at room temperature.
 10. The method of claim 1 wherein thepolymer comprises a polysiloxane, a carbon-based polymer or mixturesthereof.
 11. The method of claim 1 wherein the polymer comprisespolydimethylsiloxane (PDMS), a polyacrlyamide, a polyacrylate, apolymethacrylate or a mixture thereof.
 12. The method of claim 1 whereinthe uncured polymer is applied at a thickness in the range of about 0.1microns to about 5000 microns.
 13. The method of claim 1 wherein theuncured polymer is cured at a temperature in the range of about 23 toabout 200° C.
 14. The method of claim 1 further comprising coating themold with a hydrophobic material prior to the step of applying theuncured polymer liquid onto the mold.
 15. The method of claim 14 whereinthe hydrophobic material comprises silane or fluoronated polymercoating.
 16. The method of claim 14 wherein the hydrophobic materialcomprises a coating produced by gas plasma deposition or chemicalsurface functionalization.
 17. The method of claim 16 wherein thechemical surface functionalization uses alkoxy coupling agentscomprising silanes, titanates, zirconates and zircoaluminates.
 18. Themethod of claim 17 wherein silane comprises 1H- or2H-perfluoro-decyltrichlorosilane.
 19. The method of claim 1 wherein themold comprises PDMS.
 20. The method of claim 1 wherein the pits areprovided in an array comprising pits in evenly spaced rows, pits instaggered rows, pits of the same size, pits of the same shape, pits ofvaried sizes; and/or pits of varied shapes.
 21. The method of claim 20wherein the shape of the pits comprise polygonal, circular, oval, oroblong cross-section.
 22. The method of claim 21 wherein the polygonalcross-section comprises triangular, square, rectangular, hexagonal oroctagonal cross-section.
 23. The method of claim 1 wherein the pits havea depth in the range of from about 10 microns to about 500 microns. 24.The method of claim 23 wherein the depth is greater than 150 microns.25. The method of claim 1 wherein the pits are positioned at a distanceto create separate curvilinear cavities.
 26. The method of claim 25wherein the pits are spaced at a distance in the range of about twotimes the diameter of the opening of the pit to about ten times thediameter of the opening of the pit.
 27. The method of claim 25 whereinthe pits are spaced at a distance in the range of about 50 microns toabout 500 microns.
 28. The method of claim 1 further comprising coatingthe curvilinear cavities in the polymer substrate with a protein orbiochemicals for selective capture of a cell or a cellular by-product.29. The method of claim 28 wherein the coating is deposited byvacuum-assisted deposition and reagent exchange.
 30. The method of claim28 wherein the coating comprises antibody, receptor ligand,oligonucleotide, IgG, selectin, collagen, chemoattractant, signalingmolecule and/or fibronectin.
 31. A method of manufacturing a devicecomprising; providing a mold having one or more pits therein; applyingan uncured, liquid polydimethylsiloxane (PDMS) layer onto the mold,wherein gas is trapped in the one or more pits of the mold to create aPDMS layer/gas interface at the pit opening; allowing the uncured,liquid PDMS layer to sit on the mold; curing the uncured, liquid PDMSlayer at an elevated temperature to form a cured PDMS polymer layer,wherein the gas at the PDMS layer/gas interface forms one or more nearlyspherical microcavities in the cured PDMS polymer layer; and separatingthe cured PDMS polymer layer from the mold to obtain the device.
 32. Themethod of claim 31 further comprising coating the mold with ahydrophobic material prior to said applying the liquid PDMS layer ontothe mold.
 33. The method of claim 31 wherein the hydrophobic materialcomprises a hydrophobic silane reagent or a poly(tetrafluoroethylene).34. The method of claim 31 wherein the uncured, liquid PDMS layer iscured at a temperature of 100° C.
 35. The method of claim 31 wherein theuncured, liquid PDMS layer is cured for 1 to 2 hours.